Validation of lamin A/C staining to assess micronuclear envelope integrity. (A) Representative images of KIF18A KO cells and RPE1 cells treated with nocodazole washout stained with DAPI (DNA) and antibodies against lamin A/C and mCherry 24 h following transfection with mCherry-NLS construct. (B) Percentage of micronuclei exhibiting retainment or loss of mCherry-NLS signal as a function of continuous or incomplete nuclear membrane signal (lamin A/C). Of 140 micronuclei imaged and scored, 47 micronuclear envelopes with continuous lamin A/C signal contained mCherry signal (1 showed mCherry loss), and 87 micronuclear envelopes with incomplete lamin A/C signal experienced mCherry loss (5 retained mCherry signal). Comparison of these two criteria for the same micronuclei indicated that lamin A/C signal alone correctly predicted the integrity of the micronuclear envelope for 96% of cells (134 of 140 micronuclei). Validation data were collected from two independent experiments. (C) Representative still images of RPE1 cells expressing mCherry-H2B and NLS-EGFP that were treated with nocodazole washout to induce micronucleus formation. Cells were imaged immediately after drug washout to investigate time from micronuclear formation to time of micronuclear rupture (indicated by loss of NLS-EGFP signal from co-occurring mCherry-H2B–labeled micronucleus). White arrowheads indicate micronuclei with intact nuclear envelopes; red arrowheads indicate micronuclei after rupture. (D) Plot displaying the time from micronuclear formation to micronuclear envelope rupture for KIF18A KO and RPE1 cells treated with nocodazole washout. Mean time to micronuclear envelope rupture indicated within each condition. n = 34 (RPE1 + nocodazole washout), n = 36 (KIF18A KO). P = 0.33, ns. Data from 12 independently performed experiments. (E) Graph displaying percentage of ruptured micronuclear envelopes observed in the indicated conditions (from data in D) for micronuclei that remained within the field of view for at least 2.5 h. Statistical comparison performed using χ2 contingency test. *, P < 0.01. Noc., nocodazole.
